TY - JOUR
T1 - Expression, purification and catalytic activity of Lupinus luteus asparagine β-amidohydrolase and its Escherichia coli homolog
AU - Borek, Dominika
AU - Michalska, Karolina
AU - Brzezinski, Krzysztof
AU - Kisiel, Agnieszka
AU - Podkowinski, Jan
AU - Bonthron, David T.
AU - Krowarsch, Daniel
AU - Otlewski, Jacek
AU - Jaskolski, Mariusz
PY - 2004/8
Y1 - 2004/8
N2 - We describe the expression, purification, and biochemical characterization of two homologous enzymes, with amidohydrolase activities, of plant (Lupinus luteus potassium-independent asparaginase, L1A) and bacterial (Escherichia coli, ybiK/spt/iaaA gene product, EcAIII) origin. Both enzymes were expressed in E. coli cells, with (L1A) or without (EcAIII) a His-tag sequence. The proteins were purified, yielding 6 or 30 mg·L-1 of culture, respectively. The enzymes are heat-stable up to 60°C and show both isoaspartyl di-peptidase and L-asparaginase activities. Kinetic parameters for both enzymatic reactions have been determined, showing that the isoaspartyl peptidase activity is the dominating one. Despite sequence similarity to aspartylglucosaminidases, no aspartylglucosaminidase activity could be detected. Phylogenetic analysis demonstrated the relationship of these proteins to other asparaginases and aspartylglucosaminidases and suggested their classification as N-terminal nucleophile hydrolases. This is consistent with the observed autocatalytic breakdown of the immature proteins into two subunits, with liberation of an N-terminal threonine as a potential catalytic residue.
AB - We describe the expression, purification, and biochemical characterization of two homologous enzymes, with amidohydrolase activities, of plant (Lupinus luteus potassium-independent asparaginase, L1A) and bacterial (Escherichia coli, ybiK/spt/iaaA gene product, EcAIII) origin. Both enzymes were expressed in E. coli cells, with (L1A) or without (EcAIII) a His-tag sequence. The proteins were purified, yielding 6 or 30 mg·L-1 of culture, respectively. The enzymes are heat-stable up to 60°C and show both isoaspartyl di-peptidase and L-asparaginase activities. Kinetic parameters for both enzymatic reactions have been determined, showing that the isoaspartyl peptidase activity is the dominating one. Despite sequence similarity to aspartylglucosaminidases, no aspartylglucosaminidase activity could be detected. Phylogenetic analysis demonstrated the relationship of these proteins to other asparaginases and aspartylglucosaminidases and suggested their classification as N-terminal nucleophile hydrolases. This is consistent with the observed autocatalytic breakdown of the immature proteins into two subunits, with liberation of an N-terminal threonine as a potential catalytic residue.
KW - Asparaginase
KW - Aspartylglucosaminidase
KW - Glutathione
KW - Isoaspartyl peptidase
KW - Ntn-hydrolase
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U2 - 10.1111/j.1432-1033.2004.04254.x
DO - 10.1111/j.1432-1033.2004.04254.x
M3 - Article
C2 - 15265041
AN - SCOPUS:3242893114
SN - 0014-2956
VL - 271
SP - 3215
EP - 3226
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
IS - 15
ER -